The phosphate esters include many kinds of substances that have an important role in a body, for example, nucleotides such as adenosine-3′-triphosphate (ATP); nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA); and metabolites such as glycerol phosphoric acid and glucose phosphoric acid. It is therefore very important to measure them in the fields of environment, medicine, clinical inspection, food hygiene and biological research.
As a lot of the nucleotides such as ATP are comprised in contaminants such as microorganisms or food residuum, they are also suitable as an index for the degree of contamination in food factory or kitchen. Proteins, sugars, ATP, etc. have been used for the inspection of the contamination. However, proteins or sugars are not sufficient in sensitivity, and there are contaminants that hardly contain them. A simple luminescence method with an enzyme (luciferase) has been used for the detection of ATP. However, the reagents used in the luminescence method (luciferase and luciferin) are expensive, and an expensive apparatus is also required for this method.
Pyrophosphoric acid, a compound consisting of two phosphoric acids connected via an ester bond, has been used as an index of a reaction amount of DNA polymerase reaction in gene diagnosis and the like. As a method for the detection of pyrophosphoric acid, there are, for example, coloring and luminescence methods using precipitation reaction with calcium and magnesium ions and enzymes. However, as these methods are low in sensitivity and require complex procedures, they are not suitable for automation of a detecting apparatus or a sequential monitoring. Accordingly, the development of a simple method for their detection is needed.
Furthermore, a chemical substance comprising phosphate ester includes organic phosphorus agricultural chemicals such as tetraethyl pyrophosphoric acid. Other organic phosphorus agricultural chemicals such as parathion and malathion have a similar structure with that of phosphate ester. As these organic phosphorus agricultural chemicals are very important substances as a subject of the detection, a method or apparatus for their detection is considered very useful as well. Although gas- or liquid-chromatography has been used for their detection, they will require huge facilities and a lot of money. It is therefore required to develop a simple and inexpensive sensor and method for their detection.
As mentioned above, among the methods for the detection of phosphate ester is known the enzyme luminescence method, which requires, however, expensive reagents and apparatus. The methods for the detection of other phosphate esters depend on instrumental analysis such as HPLC. Accordingly, there is no method that can be applied to in situ detection.
On the other hand, ion-chromatography, atomic absorption methods. Etc. have been used for the detection of the concentration of phosphoric acid in a sample. However, as these methods require a sufficient purification of the sample and an expensive and big-scale apparatus, they are not suitable for the in situ detection. Although a simple ion sensor using ionophore is also known, it has a low selectivity especially for phosphoric acid, and would be difficult to use for the detection in a practically low concentration range.
Patent Document 1 discloses an invention relating to a support catalyzing chemical reaction of chemical substances, which comprises the combination of metal ions and a polymer whose structure is determined by coordination with the metal ions, or the combination of metal ions, a polymer which functions to hold the metal ions, and a polymer having electron-attracting functional groups. As one example of the application of such support is described a method for the detection of phosphate ester.
Specifically, the above patent document discloses an example for the detection of ATP, TDP and pyrophosphoric acid. However, all of these substances are detected by reduction currency (minus currency) obtained at +100 mV. This currency is derived from electrochemically reductive dissociation of a complex comprising Cu in an artificial enzyme, which is coordinated with “P” of phosphate ester.    [Patent Document 1] WO03/078060 A1 pamphlet